CX3CR1+ cells in the PNS play a key role in development of neuropathic pain in mice Jianguo Cheng, MD, PhD, Professor and Director, Cleveland Clinic Pain Medicine Fellowship Program Departments of Pain Management and Neurosciences Good afternoon! I am grateful for the opportunity to share our recent findings that “Stem cell transplantation for neuropathic pain in rats”

Disclosure Research grants from DoD (Grant ID# 10669042), NIH (NIH/NINDS R01 NS052372), and Cleveland Clinic (Anesthesiology Insititute Research Fund) We have no real or apparent conflicts of interest to report We have no conflicts of interest to report except acknowledging research grants from the DoD, NIH, and Cleveland Clinic.

Background Neuropathic Pain (NP) Pain arising from a lesion or disease of the somatosensory nervous system. Afflicting up to 18% US general population One of the most common, most debilitating, most difficult to treat, and most costly pain conditions Costs threefold higher for painful neuropathic disorder patients compared with matched control subjects. Toth C, et al. Pain Med 2009; 10:918-29. van Hecke, O., et al. Pain 155, 654-662 (2014). Finnerup, N.B., et al. The Lancet. Neurology 14, 162-173 (2015). Dworkin, R.H., et al. Pain 154, 2249-2261 (2013). Jensen MP, et al. Neurology 2007; 68:1178-82. One of the most common pain conditions, afflicting about 18% US general population One of the most debilitating, the most difficult to treat, and most costly pain conditions. Costs threefold higher for painful neuropathic disorder patients compared with matched control subjects.

Neuroinflammation Neuroinflammation in both the central nervous system (CNS) and peripheral nervous system (PNS) has been specifically implicated (1-2). Fractalkine receptor (CX3CR1) is expressed constitutively in microglia in the CNS (3). It is a unique chemokine receptor that binds only to the chemokine, fractalkine (CX3CL1). We for the first time identified a unique population of CX3CR1+ cells in the PNS We investigated the role of this population of cells in the development of neuropathic pain by utilization of CX3CR1GFP knock-in mice, CX3CR1 knock-out mice, and chimeric mice with CNS or PNS CX3CR1 deficiency. We aimed to develop an efficacious and safe therapy to treat NP. Work for several labs, including ours, have demonstrated that mesenchymal stem cell transplantation can effectively alleviate neuropathic pain in animal models. In order to facilitate the bench-to-bedside translation, we compared the therapeutic efficacy between bone-marrow derived MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs) and between intrathecal (IT) and intravenous (IV) routes of transplantation. Clark AK, et al. PNAS. 2007;104:10655-10660. Staniland AA, et al. J. Neurochemistry. 2010;114:1143-1157. Verge GM, et al. European J. Neurosci. 2004;20:1150-1160.

Hypothesis CX3CL1/CX3CR1 signaling in the PNS contributes significantly to the development of neuropathic pain after chronic constriction injury of the sciatic nerve. We hypothesized that transplantations of MSCs inhibit neuropathic pain and promote the repair of injured nociceptors in rats.

Methods Approval by the IACUC of Cleveland Clinic CX3CR1GFP/+ and CX3CR1GFP/GFP transgenic mice were used to induce neuropathic pain by CCI of the sciatic nerve. Paw withdrawal thresholds were evaluated on post-surgical days 0, 7, 14, 21 and 28. Samples of the sciatic nerve, DRG, and spinal cord of both sides for immunohistochemistry and flow cytometry examination of CX3CR1+ cells. Transgenic mice with global, CNS, or PNS CX3CR1 deficiency were used to determine the role of CX3CR1+ cells the development of neuropathic pain. Two-way ANOVA followed by paired comparisons with Bonferroni corrections The research protocols were approval by the IACUC of Cleveland Clinic. MSCs were isolated from the bone marrow or adipose tissues, enriched in culture, and characterized by flowcytometry and functional cell differentiation. MSCs were transplanted either intrathecally or intravenously. NP was induced by chronic constriction injury of the sciatic nerve and evaluated by behavioral tests to mechanical and thermal stimulation. IHC was performed to trace transplanted stem cells, monitor inflammatory changes, and evaluate the integrity of the nerve fibers by using specific biomakers. Behavioral data were analyzed by two-way ANOVA followed by paired comparisons with Bonferroni corrections. Liu L, et al. J Neurosci. Methods, 2017, in press.

The cells showed characteristic scatter plot patterns and cell markers of MSCs in flowcytometry. Scatter plots showed patterns typical of BM cell and MSCs. Also, MSCs were positive for MSC markers CD44, CD90, and CD29 and negative for hematopoietic cell markers CD45, CD31, and CD11. In contrast, the bone marrow cells were positive for all these markers.

Results Blood SC SN DRG GFP CD45 Our results demonstrate that the MSCs were able to differentiate into adipose cells containing oil droplets in the cytoplasm or osteoblast cells forming bone-like structures.

CCI significantly reduced the withdrawal thresholds to mechanical and thermal stimulation; IT transplantation of BM-MSCs dramatically reversed PWT, compared to PBS control. Please note the effects were long-lasting and low doses tended to be slightly more effective.

SN DRG SC

Conclusions We discovered a specific population of resident CX3CR1+ cells in the PNS (the sciatic nerve and DRG) which were morphologically different from blood CX3CR1+ cells and CNS CX3CR1+ microglia. PNS CX3CR1+ cells played a role in the development of neuropathic pain that was as important as microglia in the CNS. Further experiments will further clarify how this population of CX3CR1+ cells contribute to the initiation and/or maintenance of neuropathic pain.

Acknowledgements LiPing Liu, MD, PhD Yan Yin, MD Fei Li, MD Zhen Hua, MD, PhD

Thank you!